title: An activity-based probe for high-throughput measurements of triacylglycerol lipases.
abstract: Modulating the activity of lipases involved in the metabolism of plasma lipoproteins is an attractive approach for developing lipid raising/lowering therapies to treat cardiovascular disease. Identifying small molecule inhibitors for these membrane-active enzymes, however, is complicated by difficulties associated with measuring lipase activity and inhibition at the water-membrane interface; substrate and compound dynamics at the particle interface have the potential to confound data interpretation. Here, we describe a novel ELISA-based lipase activity assay that employs as "bait" a biotinylated active-site probe that irreversibly binds to the catalytic active-site serine of members of the triacylglycerol lipase family (hepatic lipase, lipoprotein lipase, and endothelial lipase) in solution with high affinity. Detection of "captured" (probe-enzyme) complexes on streptavidin-coated plates using labeled secondary antibodies to specific primary antibodies offers several advantages over conventional assays, including the ability to eliminate enzyme-particle and compound-particle effects; specifically measure lipase activity in complex mixtures in vitro; preferentially identify active-site-directed inhibitors; and distinguish between reversible and irreversible inhibitors through a simple assay modification. Using EL as an exemplar, we demonstrate the versatility of this assay both for high-throughput screening and for compound mechanism-of-action studies.
authors.affiliation: Department of In Vitro Sciences, Merck Frosst Centre for Therapeutic Research, 16711 Trans Canada Highway, Kirkland, Quebec, Canada.
authors.forename: John
authors.initials: J
authors.lastname: Tam
authors.forename: Martin
authors.initials: M
authors.lastname: Henault
authors.forename: Lianhai
authors.initials: L
authors.lastname: Li
authors.forename: Zhaoyin
authors.initials: Z
authors.lastname: Wang
authors.forename: Anthony W
authors.initials: AW
authors.lastname: Partridge
authors.forename: Roman A
authors.initials: RA
authors.lastname: Melnyk
mesh_terms: D001710:Biotin / Q000031:analogs & derivatives* / Q000737:chemistry; D019921:Biotinylation; D020134:Catalytic Domain; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D057166:High-Throughput Screening Assays; D006801:Humans; D008049:Lipase / Q000378:metabolism*; D008071:Lipoprotein Lipase / Q000378:metabolism; D013451:Sulfonic Acids / Q000737:chemistry*
chemical_list: D004791:Enzyme Inhibitors; C504173:LIPC protein, human; D013451:Sulfonic Acids; D001710:Biotin; C410382:LIPG protein, human; D008049:Lipase; D008071:Lipoprotein Lipase